ASB2 expression leads to MLL degradation and MLL target gene down-regulation during hematopoietic differentiation. (A-D) NB4 cells were treated with ATRA for 0, 48, and 72 hours to induce differentiation. The expression of ASB2 (A), the protein levels (B) and transcription levels (C) of MLL, and the expression of HOXA9 (D) were measured by RT-qPCR or Western blotting. Transcription is shown relative to the level at 0 hours. Error bars indicate SD. β-ACTIN blot indicates equal loading. (E-F) ASB2 expression vector was transfected into NB4 cells by electroporation, and the expression levels of ASB2 (E) and HOXA9 (F) were measured by RT-qPCR. Expression is shown relative to empty vector transfected control cells. Error bars indicate SD. (G-I) Expression of Asb2, Mll, and Mll target genes during 4-OHT withdrawal induced differentiation of the Hoxa9-ER cell line. (G) Heat map generated from gene expression array data collected at different time points after 4-OHT withdrawal. The Asb genes were clustered according to their expression change. Data are shown in triplicate for each time point. (H) Mll protein level in Hoxa9-ER cells was determined by Western blotting at 72 and 120 hours with or without 4-OHT withdrawal. (I) Heat map of the Mll target gene expression generated from gene expression array data as in panel G.